Interferons are cytokines produced by a variety of eukaryotic cells upon exposure to certain environmental stimuli, including mitogens, endotoxins, double stranded RNA, and viral infection. In addition to having antiviral properties, interferons have been shown to affect a wide variety of cellular functions. These effects include inhibition of cell proliferation, immune regulatory functions and activation of multiple cellular genes. Interferons (IFNs) have been classified into four groups according to their chemical, immunological, and biological characteristics: α(leukocyte), β(fibroblast), γ, and ω. IFNs are further identified by the eukaryote in which they originated, with HuIFN indicating human interferon, for instance.
HuIFN-αs are encoded by a multigene family consisting of about 20 genes; each gene encodes a single subtype of the HuIFN-α. Amino acid sequence identity among IFN-αsubtypes is generally 80-85% (Horisberger and Di Marco 1995). HuIFN-α polypeptides are produced by a number of human cell lines and human leukocyte cells after exposure to viruses or double-stranded RNA, or in transformed leukocyte cell lines (e.g., lymphoblastoid lines).
IFN-αs act through interaction with cell-surface receptors and induce the expression, primarily at the transcriptional level, of a broad but specific set of cellular genes. Several IFN-induced gene products have been used as markers for the biological activity of interferons. These include, for instance, ISG15, ISG54, IRF1, GBP, and IP10.
Individual IFN-αsubtypes have different biological activities. For instance, it was recognized early in interferon research that IFN-α1 and IFN-α2 have distinct target-cell specificities. Human IFN-α2 shows high specific activity on bovine and human cells (similar to most HuIFN-αs), whereas human IFN-α1 shows high activity only on bovine cells.
Interferon activities were first characterized in relation to viral infections, and IFN-αs have proven to be remarkably effective antiviral agents. The current definition of IFN activity units is expressed in virological terms. There are many assays known to those skilled in the art that measure the degree of resistance of cells to viruses (McNeill, 1981). These assays generally can be categorized into three types: inhibition of cytopathic effect; virus plaque formation; and reduction of virus yield. Viral cytopathic effect assays measure the degree of protection induced in cell cultures pretreated with IFN and subsequently infected with viruses. Vesicular stomatitis virus, for instance, is an appropriate virus for use in such an assay. This type of assay is convenient for screening numerous different IFNs, as it can be performed in 96-well plates (Rubinstein et al., 1981). Plaque-reduction assays measure the resistance of IFN-treated cell cultures to a plaque-forming virus (for instance, measles virus). One benefit to this assay is that it allows precise measurement of a 50% reduction in plaque formation. Finally, virus yield assays measure the amount of virus released from cells during, for instance, a single growth cycle. Such assays are useful for testing the antiviral activity of IFNs against viruses that do not cause cytopathic effects, or that do not build plaques in target-cell cultures. The multiplicity of infection (moi) is an important factor to consider when using either plaque-reduction or virus-yield assays.
Other clinically important interferon characteristics are also easily assayed in the laboratory setting. One such characteristic is the ability of an interferon polypeptide to bind to specific cell-surface receptors. For instance, some IFN-αs exhibit different cell-surface properties compared to IFN-α2b, the IFN most widely used in clinical trials. While IFN-α2b is an effective antiviral agent, it causes significant adverse side effects. Interferons that exhibit distinct binding properties from IFN-α2b may not cause the same adverse effects. Therefore, interferons that compete poorly with IFN-α2b for binding sites on cells are of clinical interest. Competitive interferon binding assays are well known in the art (Hu et al., 1993; Di Marco et al., 1994). In general, such assays involve incubation of cell culture cells with a mixture of 125I-labeled IFN-α2b and an unlabeled interferon of interest. Unbound interferon is then removed, and the amount of bound label (and by extension, bound 125I-labeled IFN-α2b) is measured. By comparing the amount of label that binds to cells in the presence or absence of competing interferons, relative binding affinities can be calculated.
Another prominent effect of IFN-αs is their ability to inhibit cell growth, which is of major importance in determining anti-tumor action. Growth inhibition assays are well established, and usually depend on cell counts or uptake of tritiated thymidine ([3H]thymidine) or another radiolabel. The human lymphoblastoid Daudi cell line has proven to be extremely sensitive to IFN-αs, and it has been used to measure antiproliferative activity in many IFN-αs and derived hybrid polypeptides (Meister et al., 1986). Use of this cell line has been facilitated by its ability to be grown in suspension cultures (Evinger and Pestka, 1981).
IFN-αs also exhibit many immunomodulatory activities (Zoon et al., 1986).
Although IFNs were first discovered by virologists, their first clinical use (in 1979) was as therapeutic agents for myeloma (Joshua et al., 1997). IFN-αs have since been shown to be efficacious against a myriad of diseases of viral, malignant, angiogenic, allergic, inflammatory, and fibrotic origin (Tilg, 1997). For instance, IFN-α is the only drug that is currently approved for treatment of hepatitis C in Europe and North America (Moussalli et al., 1998), and is the treatment of choice for chronic acute hepatitis B and AIDS-related Karposi's sarcoma. It has also proven efficacious in the treatment of metastatic renal carcinoma and chronic myeloid leukemia (Williams and Linch, 1997). Clinical uses of IFNs are reviewed in Gresser (1997) and Pfeffer (1997).
Standard recombinant techniques have become useful methods for the production and modification of IFN-α proteins (Streuli et al., 1981; Horisberger and Di Marco 1995; Rehberg et al., 1982; Meister et al., 1986; Fidler et al., 1987; Sperber et al., 1993; Mitsui et al., 1993; Muller et al., 1994; and Zav'Yalov and Zav'Yalov 1997). One such recombinant modification is the formation of hybrid IFN molecules. Hybrid IFNs contain fragments of two or more different interferon polypeptides, functionally fused together. The first IFN-αhybrids were designed to study molecular structure-function relationships. Much research has since been directed toward the production of hybrid IFNs that combine different biological properties of the parental proteins. Some hybrid IFNs display biological activity that is significantly different from that of both parent molecules (Horisberger and Di Marco 1995). For instance, certain early IFN-α/IFN-α hybrids acquired the novel property of very high activity on mouse cells (Streuli et al., 1980; Rehberg et al., 1982).
The techniques used by researchers to generate hybrid IFN polypeptides have evolved through time (Horisberger and Di Marco 1995). Early researchers took advantage of the presence of naturally occurring restriction endonuclease (RE) cleavage sites within IFN-encoding sequences to piece together homologous coding fragments. (See, for instance, U.S. Pat. No. 5,071,761 “Hybrid Interferons”). Though convenient, this was a limited method in that only so many of such pre-existing RE sites occurred in each IFN coding sequence. In addition, the location of each restriction site was fixed, making the possible combinations relatively small. More recently, researchers have used PCR amplification to create specific desired nucleic acid fragments, thereby gaining the ability to piece together new pieces of different IFNs (Horton et al., 1989).
A number of U.S. patents discuss various hybrid IFNs, how to produce them, and how to use them to treat patients. Many such patents relate to inter-group (multi-class) hybrid IFNs, wherein portions of the final hybrid are taken from at least two different interferon classification groups (e.g., α and β). For instance, U.S. Pat. No. 4,758,428 (“Multiclass hybrid interferons”) describes the multi-class hybrid IFN HuIFN-α1 (1-73)/HuIFN-β1(74-166), and its use in pharmaceutical compositions to treat viral infections and tumorous growths in animal patients. Another such patent (U.S. Pat. No. 4,914,033 “Structure and properties of modified interferons”) discloses the making of constructs that encode hybrid interferons comprising amino- and carboxy-terminal fragments of HuIFN-β fused to an internal sequence (amino acid residues 36-46) of a HuIFN-α. This patent also discloses the purification of the encoded hybrid IFN polypeptide and its use in pharmaceutical formulations.
Intra-group hybrid interferons (e.g., α1/α8 hybrids) have also been described. U.S. Pat. No. 5,071,761 (“Hybrid interferons”) provides a good example of such intra-group hybrids. This patent discloses the construction, purification, use, and pharmaceutical preparation of various fusions hybrids between HuIFN-α1 and HuIFN-α8, where as many as four distinct IFN-α fragments have been used to construct the fusion. The construction, purification, and use of similar IFN-α hybrids to treat animal patients are disclosed in U.S. Pat. No. 5,137,720 (“Antiviral combination, and method of treatment”).
It is to such engineered, recombinant intra-group hybrid interferon molecules that the present invention is directed.